The complexity of multi-cellular organisms characterized by cell specialization and sophisticated tissue and organ architecture is possible because of an efficient indexing system that keeps track of which sections of the genome are active or repressed, according to cell type and developmental phase. This "epigenetic" information (independent of DNA sequence) not only needs to be accurately established, but must also be transmitted to the cell's progeny. This must be achieved through a number of molecular mechanisms, many of which impact the structure of chromatin, particularly histones. Key to cell identity is the establishment and maintenance of "facultative heterochromatin", i.e. the silencing of specific genes in certain cell lineages through the formation of chromatin structure(s) repressive to transcription. The goal of this proposal is to expand our previous studies on the molecular mechanisms underlying facultative heterochromatin. How is selectivity achieved? What chromatin structures contain epigenetic information? How is this information transmitted through cell division? We will answer these and other questions through traditional and innovative biochemical approaches. In aim 1, we will expand our studies on PRC2, a central chromatin regulator on which we have accumulated much molecular information in the last years. We will study further: the function of EZH1, an EZH2 homolog with less pronounced methyltransferase activity;the effects of optional PRC2 complex subunits in cell differentiation and cancer;and the role of two novel EZH2 phosphorylation sites on PRC2 activity, chromatin localization, and interaction with non-coding RNAs (ncRNAs). In aim 2, we will employ TIRF microscopy and state-of-the-art mass spectrometry to determine whether sister histones within the same nucleosome carry any symmetrical post-translational modification, which would be strong candidates for "true" epigenetic marks as they may be equally segregated upon DNA replication. In aim 3, we will expand our studies on MBT proteins, an understudied family of chromatin binders;in particular we will investigate the biochemical and cellular function of SFMBTs and SCMH1/L2, which contain MBT repeats and were genetically classified as Polycomb group genes. The role of Pr-SET7 in preserving chromatin structure, protecting from or signaling DNA damage, its interaction with PCNA and its interaction with the cognate histone reader, L3MBTL1, will be investigated in aim 4, as well as the potential for this interaction to transmit epigenetic information. In aim 5, we will continue and expand our studies on SirT3, a fascinating Sirtuin with a dual role in mitochondrial function and regulation of nuclear transcription. We will explore this connection, as well as post-translational modifications and interactors of SirT3. PUBLIC HEALTH RELEVANCE: A key to development of multi-cellular organisms is the maintenance of distinct cell identities resulting from different patterns of gene expression;cells keep track of these by assembling unused parts of the genome in a repressive structure called "heterochromatin". When this process is compromised, cells can acquire a new identity, often transforming into cancer cells. The aim of this proposal is to better understand the molecular mechanisms involved in this process, and their role in disease such as cancer.